Avian influenza virus vaccine and a method for preparing

ABSTRACT

The present invention relates to an avian influenza vaccine of plant origin, prepared by transforming a plant with a surface protein of avian influenza virus, hemagglutinin (HA) or neuraminidase (NA), and a method for preparing the same. Further, the present invention relates to an oral vaccine for preventing avian influenza virus, of which mass-production conditions are established, and thus the produced transgenic plant can be more easily, safely, and economically used as a fodder additive. Furthermore, the present invention relates to a reagent for diagnosing avian influenza virus infection, prepared by isolating and purifying a recombinant antigen protein from the transgenic plant.

TECHNICAL FIELD

The present invention relates to a vaccine for preventing avian influenza virus infection. More particularly, the present invention relates to an oral vaccine, in which the surface protein of avian influenza virus, hemagglutinin (HA) or neuraminidase (NA) is expressed in transgenic plants, and to a method using the same.

BACKGROUND ART

In recent years, avian influenza outbreaks have occurred worldwide and affect humans to cause severe disease conditions, thereby attracting much attention. In particular, the spread of avian influenza in the poultry industry causes devastating economic losses. Accordingly, there is an urgent need to develop a vaccine to protect birds against avian influenza.

The avian influenza virus is an RNA virus belonging to the family Orthomyxoviridae, of which the genome is split to 8 RNA segments and easily modified by recombination. The influenza virus is characterized by having multiple serotypes, and no cross protection occurs between different serotypes. Currently, the virus is largely classified into three types (A, B, and C) according to antigenic differences in the nucleoprotein and the matrix protein, and all of the animal influenza viruses belong to type A.

The influenza virus type A has two proteins, hemagglutinin (HA) and neuraminidase (NA) on its surface. Hemagglutinin (HA) has a binding site with sialic acid that is present in host bronchial epithelial cells, and enters the nucleus via receptors to produce new influenza virus particles. Neuraminidase (NA) cleaves the linkage between sialic acid and viral HA to destroy the receptors, resulting in preventing the accumulation of newly produced viral particles on the infected cell membrane.

Hemagglutinin (HA) is classified into 16 HA subtypes (H1˜H16), and neuraminidase (NA) into 9 NA subtypes (N1˜N9) according to the antigenic properties, which potentially allows 144 various combinations of the influenza virus type A. Among them, the subtypes H5 and H7 are known to be pathogenic to birds, and the subtypes H1, H2, and H3 are known to cause influenza in humans. It has been known that avian influenza virus does not infect any animal, other than avian and swine species. However, an H5N1 avian influenza virus was isolated from patients in Hong Kong in 1997, which confirmed the possibility of human infection by the avian influenza virus. The human infection is thought to be caused by highly pathogenic viruses, which are generated by genetic mutation of avian influenza virus and human influenza virus, when they simultaneously infect human. These highly pathogenic viruses may transmit from person to person, and the risk of pandemic is increasing. Therefore, there is a need for developing a safe vaccine for birds, which is a host for avian influenza virus.

However, the avian influenza virus has a variety of serotypes, and there is little cross-immunity among the subtypes. Thus, it is hard to prevent the infection by other serotypes. Further, since the avian influenza viruses are highly apt to undergo mutation, there is no effective vaccine for preventing the avian influenza. Currently, the most effective prevention method is washing with antiseptic agents, and parenteral vaccination with inactivated influenza virus vaccine or a recombinant fowl pox virus vaccine. However, such methods can be performed, even after being infected with avian influenza and examination of the subtype of virus. Further, these vaccines may reduce the amount of virus in feces from infected poultry, but hardly prevent the spread of the disease and also incur high cost.

As alternatives, oral vaccine and edible vaccine that are directly applied to mucous membranes are suggested, and many studies have been conducted thereon, for example, oral administration of avian influenza virus itself (John M. Crowford et al., Avian disease, (1998), 42(3):486-96)) and oral spray immunization with influenza virus. However, for the preparation of these vaccines, attenuated virus or viral proteins expressed in bacteria are used, which are different from those expressed in mammals.

Meanwhile, a plant has a eukaryotic protein synthesis pathway, in which post-translational modifications being essential for mammals occur (Cabanes-Macheteau et al., Glycobiology 9.365-372 (1999)). Thus, plants are able to produce proteins similar to those expressed in mammals. For that reason, much of the focus has been placed on the production of desired proteins using transgenic plants.

The present inventors have expressed an HA or NA protein specific to avian influenza virus in transgenic plants using their properties, and established the condition for mass-production to develop an avian influenza vaccine of plant origin, which is a safe and economical mucosal vaccine, thereby completing the present invention.

DISCLOSURE Technical Problem

It is an object of the present invention to provide an oral vaccine for preventing avian influenza virus infection using transgenic plants.

Technical Solution

In order to develop an oral vaccine for preventing avian influenza virus infection, the present inventors have transformed each of HA and NA proteins of avian influenza virus into hairy roots of Korean melon, and developed a mass production system for producing the recombinant hairy roots as a fodder additive, thereby completing the present invention.

ADVANTAGEOUS EFFECTS

The transgenic plant according to the present invention is an oral vaccine for preventing the infection of high or low pathogenic avian influenza virus by using as a fodder additive for wild birds and poultry, in which the hairy roots transformed with HA or NA of avian influenza virus are mass-produced, and the high expression of anti-HA antibody is confirmed in serum from animal treated with the transgenic hairy roots. The transgenic plant as vaccine can be produced in a large amount with a low production cost, as compared to a known vaccine, in particular, can be used as the oral vaccine to achieve excellent effects such as convenience of immunization and induction of mucosal immunity.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating a pBSNB22101 vector for the construction of the vector for plant transformation according to the present invention;

FIG. 2 is the result of PCR for the detection of Tobacco Etch Virus (TEV) leader sequence;

FIG. 3 is the result of PCR for the detection of hemagglutinin (HA) and neuraminidase (NA), which are surface proteins of avian influenza virus;

FIG. 4 is a diagram schematically illustrating the construction process of a pBSNB-TEV vector, which is the vector for plant transformation according to the present invention;

FIG. 5 is the result of PCR for the confirmation of TEV cloning into pBSNB22101;

FIG. 6 is a diagram illustrating the HA and NA cloning into pBSNB-TEV;

FIG. 7 is the result of electrophoresis showing the size and concentration of HA and NA inserts and pBSNB22101-TEV vector before ligation;

FIG. 8 is a photograph showing the insertions of HA and NA at the positions of 1695 bp and 1350 bp, in which each of the ligated plasmid DNAs is digested with Nru I/Hind III (HA) and Kpn I/Hind III (NA), used upon ligation;

FIG. 9 shows partial base sequence comparison of a cloned HA gene and a template HA gene;

FIG. 10 shows partial amino acid sequence comparison, in which an amino acid sequence translated from the HA base sequence is aligned with the amino acid sequence of the template HA protein;

FIG. 11 shows partial base sequence comparison of a cloned NA gene and a template NA gene;

FIG. 12 shows partial amino acid sequence comparison, in which an amino acid sequence translated from the NA base sequence is aligned with the amino acid sequence of the template NA protein;

FIG. 13 is the result of PCR of clones #1˜5 for the selection of transformed Agrobacteria to be used for plant transformation;

FIG. 14 is a photograph showing the HA transgenic hairy root;

FIG. 15 is the result of screening the HA transgenic hairy roots by HA Ag ELISA;

FIG. 16 is the result of PCR for the detection of HA genomic DNA from the transgenic hairy roots;

FIGS. 17 a) and b) are diagrams illustrating the location of primer sequences used for RT-PCR of HA and NA;

FIG. 18 a) and b) are the results of RT-PCR of HA and NA, in which the amount of RNA transcript is indicated by the band strength;

FIG. 19 a) and b) are photographs showing the production of HA and NA recombinant proteins by a BL21 strain;

FIG. 20 is the result of Western blot showing the presence of HA protein from the hairy root extract;

FIG. 21 is the result of Western blot showing the expression of HA protein in the air-dried hairy root;

FIG. 22 is the result of HA glycosylation pattern analysis using PNGase F;

FIG. 23 is a photograph showing the selection of optimal medium for mass-production of HA-20 hairy root;

FIG. 24 is a photograph showing the yield of hairy root according to pH levels;

FIG. 25 is a photograph showing the selection of optimal sucrose concentration in the predetermined pH and media;

FIG. 26 is a photograph showing the applicability of selected liquid culture conditions to an 18 L mass-production system;

FIG. 27 is the result of quantifying the expression of HA protein in the freeze-dried transgenic hairy root powder;

FIG. 28 shows an immunization schedule;

FIG. 29 is a graph showing the result of HA specific serum IgG induction in the mice that are orally administered with the HA transgenic hairy root; and

FIG. 30 is a graph showing the result of HA specific fecal IgA induction in mice that are orally administered with the HA transgenic hairy root.

BEST MODE

Accordingly, the present invention is characterized in that a plant is transformed with antigen proteins of avian influenza virus, and the transgenic plant is mass-produced, thereby being used for a fodder additive as an oral vaccine for preventing avian influenza virus infection. Hereinafter, the present invention will be described in detail with reference to embodiments.

In one aspect, the present invention provides a novel recombinant expression vector, in particular, being suitable for plant transformation.

There are many known vectors for plant transformation. However, in the present invention, a novel vector for plant transformation is constructed, thereby expressing the surface proteins of avian influenza virus in plants in a high yield.

The vector for plant transformation comprises (a) a replication origin operating in plant cells, (b) a promoter promoting transcription in plant cells, (c) a polycloning site capable of introducing a structural gene sequence encoding a foreign protein, (d) a polyadenylation signal sequence that operates in plant cells to form polyadenosine at a 3′ end of RNA, and further may comprise an antibiotic resistance gene as a reporter gene.

In a preferred embodiment, the vector for plant transformation comprises cauliflower mosaic virus 35S promoter and 35S terminator, and further comprises a polycloning site for introducing a sequence that encodes a foreign protein to be expressed and TEV (Tobacco etch virus) leader sequence to increase expression level. Additionally, the vector for plant transformation further comprises an antibiotic resistance gene as a reporter gene. According to the preferred embodiment of the present invention, a vector for plant transformation, pBSNB-TEV is constructed, which is shown in FIG. 4.

According to the present invention, a nucleic acid sequence encoding the antigen protein of avian influenza virus is introduced into the vector for plant transformation as described above, so as to prepare a recombinant expression vector for expressing antigen proteins of avian influenza virus in plants. The antigen protein of avian influenza virus is preferably an HA or NA protein of avian influenza virus, but is not limited thereto. In addition to HA and NA, nucleic acid molecules that encode proteins capable of inducing cellular immune response, such as nucleoprotein (NP), RNA polymerase protein PB2 (polymerase basic protein 2), and polymerase acid protein (PA), can be used for the preparation of the recombinant expression vector for expressing antigen proteins of avian influenza virus.

In the preferred embodiment of the present invention, vectors having HA and NA genes were provided by National Veterinary Research & Quarantine Service to produce recombinant HA and NA proteins of avian influenza virus as antigen proteins. Specifically, the HA gene is isolated from a low pathogenic avian influenza virus, H5N3 strain, which is disclosed in Korean Patent No. 693858, and the NA gene is isolated from a highly pathogenic avian influenza virus A/CK/Korea/ES/03[H5N1](CK/Kr/ES/03), which was first isolated in Korea in 2003. pCR2.1-HA and pCR2.1-NA vectors, which contain each fragment of the above genes, were provided by National Veterinary Research & Quarantine Service, and PCR was performed to amplify the gene fragments. The gene fragments were introduced to the above vector for plant transformation to construct a recombinant expression vector for expressing antigen proteins of avian influenza virus. The construction process of the vector is described in detail in FIG. 4 and Examples 1.1 and 1.2.

In another embodiment, the present invention provides a method for preparing an avian influenza virus vaccine of plant origin, comprising the step of transforming plants using the recombinant expression vector for expressing antigen proteins of avian influenza virus.

As mentioned above, recombinant protein expressed in plants undergoes post-translational modifications by the same processes as are found in mammalians. Further, plants can be produced in a large amount under specific conditions, thereby being used for fodder additives. Accordingly, the vaccine of the present invention is excellent in terms of efficacy, safety, economy, and convenience, thereby being industrially useful.

Examples of the plant transformation method include a method for transporting a foreign DNA to plant cells using a biological vector such as Agrobacterium, and a method for directly transporting a foreign DNA to plant cells as transporting DNA-coated metal microparticles to plant cells (Hansen and Wright, Trends Plant Sci. 4; 226-231, 1999). Currently, the plant transformation method using a plant pathogen, Agrobacterium is generally used. Agrobacterium has an ability to transfer T-DNA of its Ti (Tumor-inducing) or Ri (root-inducing) plasmid DNA to the genome of infected plant cells. Accordingly, the recombination of a desired gene with T-DNA is performed, and then bacteria having the recombinant are co-cultured with a plant, thereby achieving the plant transformation.

In a specific embodiment of the present invention, an Agrobacterium rhizogenes strain having a Ri plasmid is transformed with the recombinant expression vector for expressing antigen proteins of avian influenza virus, and then the transformed Agrobacterium strain is used to transform a plant, in particular, cotyledon and hypocotyl of Korean melon, so as to induce transformed hairy roots of Korean melon (see Example 2). To confirm whether the genomic DNA of the hairy root is integrated with the gene encoding HA or NA, which is a surface protein of recombinant avian influenza virus, genomic DNA PCR was performed, and the expression is measured by RT-PCR, Western blot, and ELISA using anti-HA antibody (Example 3). Further, it was confirmed that the expressed HA or NA protein was glycosylated via post-translational modification (Example 4).

In order to confirm whether the transgenic plant expressing the recombinant HA or NA protein can be used as a fodder additive, the hairy roots were subjected to air-drying, followed by oral administration to mice. Sera and feces were collected from the mice, and subjected to ELISA to determine the antibody titer. As a result, HA-specific antibodies were more highly induced than in a negative control administered with the hairy roots that were transformed with Agrobacterium rhizogenes strain having an empty vector (see Example 8 and FIGS. 29 and 30). Accordingly, it was found that the transgenic plant itself according to the present invention can be used as an oral vaccine for preventing avian influenza virus infection.

In still another embodiment, the present invention provides an avian influenza virus vaccine, comprising the plant transformed with the recombinant expression vector for expressing antigen proteins of avian influenza virus. In this embodiment, even though the vector was transformed into Korean melon only, and the hairy roots therefrom was confirmed to be used as an oral vaccine, the plant to be used in the present invention is not limited thereto. Namely, any plant may be used, as long as it can induce hairy roots by transformation with the recombinant expression vector for expressing antigen proteins of avian influenza virus. Examples thereof include rice (Oryza sativa), barley (Hordum sativum), wheat (Triticum aestivum or Triticum vulgare), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), cucumber (Cucumis sativus), and Korean melon (Cucumis melo).

In still another embodiment, the present invention provides a method for mass-producing the transgenic plant. As mentioned above, the transgenic plant itself according to the present invention may be used as a fodder additive. In accordance with this, the transgenic plant itself can act as an oral vaccine. In order to use the transgenic plant as a vaccine, a prerequisite is to establish a method for economically and stably producing the transgenic plant in a large amount. The present inventor established the conditions for mass-production of the transgenic plant.

Specifically, the production method is a method capable of mass-producing the transgenic hairy root in a bioreactor, and the optimal conditions such as type of medium, pH, and sucrose concentration was established. It can be seen that the transgenic plant according to the present invention grows well in MS or B5 medium (see FIG. 23), and better at neutral pH from pH 7 to 8 than at a known pH 5.8 (see FIG. 24). Further, in respect to sucrose concentration as an energy source, there is no difference between 15 g/L and 30 g/L. Therefore, the concentration of 15 g/L was determined in terms of the production cost (see FIG. 25).

According to such production method, the oral vaccine against avian influenza virus can be prepared with ease and at low cost.

In still another embodiment, the present invention provides a reagent for diagnosing the infection of avian influenza virus, prepared by isolating and purifying the antigen protein of avian influenza virus from the transgenic plant.

As a primary object, the present invention tried to provide an oral vaccine, by using the plant transformed with the antigen protein of the avian influenza virus as a fodder additive. Additionally, a reagent for diagnosing the infection of avian influenza virus can be prepared by easily isolating and purifying a large amount of antigen protein from the transgenic plant. Accordingly, in still another embodiment, the present invention further provides a method for preparing a diagnostic reagent for avian influenza virus infection, comprising the step of isolating the antigen proteins of avian influenza virus from the transgenic plant. According to the present embodiment, the antigen protein of avian influenza virus is isolated and purified from the transgenic plant, and then subjected to reaction with serum from bird, human or the like, so as to detect the infection of avian influenza virus.

Hereinafter, the present invention will be described in detail with reference to Examples. However, these Examples are for the illustrative purpose only, and the invention is not intended to be limited by these Examples.

MODE FOR INVENTION 1. Construction of Vector for Plant Transformation Containing Antigen Gene 1.1 Development of Vector for Plant Transformation

A vector for plant transformation, containing comprising cauliflower mosaic virus 35S promoter, polycloning site, TEV (tobacco etch virus) leader sequence, and 35S terminator, was prepared as follow.

First, pCAMBIA 2300 (CAMBIA, Australia) was digested with a restriction enzyme, Hind III and dephosphorylated with calf intestine phosphatase. Then, a pDPG165 plasmid (Kausch et al., 2001) was digested with Hind III, and ligated into the digested pCAMBIA 2300 to prepare a pBS221 plasmid. The restriction sites in the pBS221 plasmid (BamH I, Kpn I, Xba I) were digested with each restriction enzyme, and polymerization was performed with T7 polymerase, respectively.

Meanwhile, in order to insert the polycloning site into pBI221 (Clontech, USA), pBI221 was digested with Sac I and Xba I, ligated with a synthetic primer 1, oligonucleotide (SEQ ID NO. 1: 5′-GACGCGTGAGCTCGGTACCTCGCGAGGATCCTCTAGAGTCGACCTGCAGG CATGCAAGCTTG-3′, and subjected to polymerization with T7 polymerase, followed by blunt end ligation. In pBI221 containing the oligonucleotide, a fragment containing CaMV 35 promoter, polycloning site and NOS terminator was amplified using the oligonucleotide, primer 2 (SEQ ID NO. 2: 5′-CGGAATTCAGATTAGCCTTTTCAATTTCAGAAAG-3′, and primer 3 (SEQ ID NO. 3: 5′-CGGAATTCGATCTAGTAACATAGATGACACC-3′ by PCR (PCR condition: 95° C. 5 min, one cycle; 94° C. 30 sec, 60° C. 30 sec, 72° C. 2 min, 30 cycles; 72° C. 7 min, 1 cycle), followed by digestion with a restriction enzyme, EcoR I. Then, the fragment was cloned into pBS221 to prepare a pBSNB22101 vector (FIG. 1).

Further, to increase expression level, TEV (Tobacco etch virus) leader sequence was amplified using pRTL2 (Carrington and Freed et al., J. Virol. 64, 1990) as a template, a primer 4 (SEQ ID NO. 4: 5′-CGACGCGTAAATAACAAATCTCAACACAACATATAC-3′, and a primer 5 (SEQ ID NO. 5: 5′-CGGGATCCAAATAACAAATCTCAACACAACATATA-3′ by PCR (PCR condition: 95° C. 5 min, one cycle; 94° C. 30 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles; 72° C. 7 min, 1 cycle), followed by digestion with Mlu I and Kpn I (FIG. 2). The TEV fragment having a size of 142 bp was inserted into the restriction sites, Mlu I and Kpn I of pBSNB22101. The ligated vector was transformed into DH5α and cultured on plates with kanamycin (Km) at 37° C. Since the pBSNB22101 vector had a kanamycin resistance gene, colonies were formed on the plates. On the next day, colonies formed on plates were selected, and cultured in 3 ml of LB liquid media having kanamycin at 37° C., followed by miniprep. Enzyme digestion was performed to confirm the presence of TEV. As shown in FIG. 5, a band of 142 bp corresponding to TEV was found. The vector was designated as pBSNB-TEV, constructed as the vector for plant transformation. The entire process of constructing the vector is shown in FIG. 4.

1.2 Development of Vector for Plant Transformation Containing HA and NA Genes of Avian Influenza Virus

In connection with the production of the vector for plant transformation containing HA and NA genes of avian influenza virus, vectors containing HA and NA genes were provided by National Veterinary Research & Quarantine Service, so as to produce HA and NA recombinant proteins.

The HA gene contained in the vector is a gene obtained from low pathogenic virus A/wild bird feces/Korea/CSM2/02 [H5N3](Wbf/Kr/CSM2/02), and viral RNA was extracted using an RNeasy mini kit (QIAGEN, Valencia, Calif.). The method for obtaining the HA gene is the same as in published paper (Lee, C. W., D. L. Suarez, T. M. Tumpey, H. W. Sung, Y. K. Kwon, Y. J. Lee, J. G. Choi, S. J. Joh, M. C. Kim, E. K. Lee, J. M. Park, X. Lu, J. M. Katz, E. Spackman, D. E. Swayne, and J. H. Kim. Characterization of highly pathogenic H5N1 avian influenza A viruses isolated from South Korea. J. Virol. 79:3692-3702. 2005.), described as follows: The HA gene fragment of about 1.7 kb was amplified using an HA-F forward primer (SEQ ID NO. 22: 5′-TATTCGTCTCAGGGAGCAAAAGCAGGG G-3′, an HA-R reverse primer (SEQ ID NO. 23: 5′-ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3′, and a One-Step RT-PCR kit (QIAGEN). RNA extraction was performed using an RNAeasy mini kit (Qiagen) and PCR was performed using a onestep RT-PCR kit (Qiagen) (PCR condition: cDNA synthesis (50° C. 30 min, 95° C. 15 min), denaturation (94° C., 45 sec), annealing (53° C., 15 sec), and extension (72° C., 1 min and 30 sec) for 30 cycles, and further reaction (72° C. for 5 min). The amplified gene fragment was subjected to sequencing analysis, and the result is the same as SEQ ID NO. 24. The amplified HA fragment was cloned into a pCR2.1 vector using a TA cloning kit (Invitrogen, N.Y., USA). The cloned vector, pCR2.1-HA was provided as a template of HA gene.

Meanwhile, the NA gene is a gene obtained from a highly pathogenic virus A/CK/Korea/ES/03[H5N1](CK/Kr/ES/03), which is first isolated in Korea in 2003, and viral RNA was extracted using an RNeasy mini kit (QIAGEN, Valencia, Calif.). As in published paper (Lee et al. J. Virol. 79:3692-3702. 2005.), the NA gene was amplified using a One-Step RT-PCR kit (QIAGEN), and then cloned into the pCR2.1 vector using a TA cloning kit (Invitrogen, N.Y., USA). The cloned vector, pCR2.1-NA was provided as a template of NA gene. Sequence information for the NA gene of CK/Kr/ES/03 strain has been deposited in GenBank (ACCESSION No. AY676043), which is represented by SEQ ID NO. 25.

To amplify the HA gene in the provided vector, pCR2.1-HA, a primer 6 (SEQ ID NO. 6:5′-GGGTCGCGAATGGAGAAAATAGTGCTTCTTCTTG-3′ and a primer 7 (SEQ ID NO. 7: 5′-CCCAAGCTTTTAAATGCAAATTCTGCATTGTAACG-3′ were designed. The NA gene in the provided vector, pCR2.1-NA has a size of about 1350 bp, and to amplify the NA gene, a primer 8 (SEQ ID NO. 8: 5′-CGGGGTACCATGAATCCAAATCAGAAGATAATAAC-3′ and a primer 9 (SEQ ID NO. 9: 5′-CCCAAGCTTCTACTTGTCAATGGTGAATGGC-3′ were prepared.

The genes coding for HA and NA were amplified using the primers by PCR (condition: 95° C. 5 min, 1 cycle; 94° C. 30 sec, 60° C. 30 sec, 72° C. 1 min, 40 cycles; 72° C. 7 min, 1 cycle), and the PCR product was subjected to electrophoresis. As a result, bands of 1695 bp and 1350 bp corresponding to HA gene and NA gene were found (FIG. 3).

Next, to ligate each of the amplified HA and NA genes into the vector for plant transformation, pBSNB-TEV prepared in Example 1.1, the amplified HA gene was digested with Nru I and Hind III, and the amplified NA gene was digested with Kpn I and Hind III (FIG. 6). Before ligation, the size of the insert and vector and their concentrations were determined by agarose gel electrophoresis (FIG. 7). The vector and insert were ligated in a ratio of 1:3 at 16° C. for 30 minutes. Two ligated plasmids were transformed into DH5α by heat shock at 42° C., and then cultured on kanamycin plates at 37° C. The next day, the selected colonies were cultured in 3 ml of LB media at 37° C. overnight. After performing miniprep, the obtained plasmid DNAs were digested with Nru I/Hind III (HA) and Kpn I/Hind III (NA) to confirm the insertions of HA and NA.

Inserts of 1695 bp and 1350 bp corresponding to HA and NA genes were found in each DNA obtained from four clones by miniprep. Among them, each of clone NO. 1 (#1) was selected, and then restriction enzyme analysis was performed to confirm whether the inserted DNAs are HA and NA genes (FIG. 8). Further, sequencing analysis was performed to confirm whether the base sequences of cloned HA and NA (SEQ ID NOs. 10 and 12) correspond to the base sequences of template HA and NA. It was also confirmed that amino acid sequences (SEQ ID NOs. 11 and 13) translated from the base sequences are correct (see FIGS. 9 to 12).

Accordingly, the vector for plant transformation, pBSNB-TEV cloned with HA gene is designated as pBSNB-TEV-HA#1-1, and the vector for plant transformation, pBSNB-TEV cloned with NA gene is designated as pBSNB-TEV-NA#1-1.

1.3 Preparation of Agrobacterium Transformant for Plant Transformation

pBSNB-TEV-HA#1-1 and NA#1-1 prepared in Example 1.2 were transformed into an Agrobacterium rhizogenes strain, K599 by a freeze-thaw method (Glevin et al., Plant molecular biology. Kluwer academic Publishers A3/7, 1988).

First, competent K599 cells were prepared, and mixed with the vectors. The heat shock was applied at 37° C. for 5 min. Then, 1 ml of antibiotic-free YEP media (10 g/L yeast, 5 g/L NaCL, and 10 g/L peptone) was added thereto, and cultured at 28° C. for 4 hrs. Subsequently, the cells were cultured on YEP plates (10 g/L yeast, 5 g/L NaCL, 10 g/L peptone, 15 g/L agar) containing kanamycin (Km)(100 ug/ml) at 28° C. for 2 days. Colonies were selected from the plates, and cultured in 3 ml of YEP media at 28° C. Genomic DNA was extracted from the transformed K599, and PCR was performed to confirm the integration into genomic DNA. PCR was performed at an annealing temperature of 60° C. for 35 cycles. Then, the product was subjected to agarose gel electrophoresis to confirm each band. As shown in FIG. 13, each band of 1695 bp and 1350 bp corresponding to HA and NA was found. Agrobacteria transformed with each clone NO. 1 (#1) of HA and NA were selected to use for plant transformation.

2. Transformation of Inserted HA and NA Genes into Hairy Root

The Agrobacteria K599 transformed with HA and NA, prepared in Example 1.3, were transformed into cotyledon and hypocotyls of Korean melon. The sterilized seeds of Korean melon were sown and germinated for 4 days under sterile conditions. Then, the stalks were removed from 4-day-old seeds and the young leaves were wounded. These wounded leaves were transformed with Agrobacterium rhizogenes strain, K599, which was transformed with pBSNB-TEV-HA#1-1 and NA#1-1 plasmids.

Each transformed K599 strain with HA and NA was cultured in 30 ml of YEP media containing kanamycin (Km) (100 ug/ml) at 28° C., and then centrifuged at 4,000 rpm for 15 min to discard supernatant. The pellets were resuspended with 30 ml of dilution of selection media (pH 5.8) containing Murashige-Skoog (MS0) media (with vitamin) (Duchefa, Netherlands), 3.0% sucrose, 1 mg/l BA (benzyladenine), 0.1 mg/l NAA (napthalene acidic acid), 0.3% phytase, 300 mg/l kanamycin and 500 mg/l carbenicillin, and sterilized water (1:2). Optical density (O.D.) values were measured, and dilution was performed with 50% MS0 to give the total OD value of 0.4. The wounded leaves of Korean melon were mixed with the above prepared solution for 15 min. Water was completely removed from the leaves using sterilized 3M paper. Then, the leaves were transferred to MS0 plates, cultured at 22° C. for 2 days in the dark, and then transferred to selection media, MS (Carbenicillin500, KM300).

The media were observed every day to discard the leaves infected with Agrobacterium rhizogenes, and other leaves in the same media were transferred to fresh selection media until hairy roots were induced. After 1˜2 weeks, the formation of hairy root was observed. One root of them was cut, and cultured in MS media containing kanamycin to select the transferred gene and carbenicillin to prevent the overgrowth of Agrobacterium rhizogenes. As shown FIG. 14, the hairy roots transformed with HA were obtained, and in the same manner, the hairy roots transformed with NA were obtained. Further, as a negative control, a transgenic plant K599 transformed with a K599 strain, which was not transformed with the plasmids, was obtained. The compositions of the MS0, MS0 and MS media are shown in the following Table 1.

TABLE 1 MS0 % MS medium (with Vitamin) 30 g/l sucrose, pH 5.8 MS0 plate % MS medium (with Vitamin) 30 g/l sucrose, pH 5.8 0.3% agar MS (Cb500, Km300) % MS medium (with Vitamin) plate 30 g/l sucrose, pH 5.8 0.3% agar Cabenicillin 500 ug/ml Kanamycin 300 ug/ml

3. Confirmation of Antigenic HA Protein Expression in Transgenic Hairy Root 3.1 HA Ag ELISA

In order to confirm the HA expression in 30 transgenic hairy root lines and compare the expression level between the lines, ELISA was performed using HA antibody. First, each hairy root was collected from media, and the agar ingredient was removed from the hairy root. The hairy root was ground into a fine powder with liquid nitrogen in a mortar and pestle. A lysis buffer (50 mM Tris-HCl, 400 mM NaCl, 1 mM EDTA, 5% glycerol, 1% Triton X-100, pH 7.5) containing a general protease inhibitor (Sigma, ST. LOUIS, USA) was added to the hairy root powder, and the protein was extracted by centrifuge at 4° C. Then, the concentration of the total extract was determined by a Bradford assay. 60 μg of the total extract was diluted with the lysis buffer to a total volume of 110 μl, and then loaded on the plate coated with monoclonal anti-HA antibody. After incubation at 37° C. for 1 hr, the plate was washed with a washing buffer 5 times. Then, monoclonal anti-HA-HRP was added thereto, and incubated at 37° C. for 30 min. After washing with the washing buffer 5 times, the substrate was added, and color-developed. After 10 min, a blocking solution was added to block the reaction, and absorbance was measured at 450 nm using a dual beam spectrophotometer. The expression levels in 30 transgenic hairy root lines were different from each other, and the cut-off level was set at the OD value of 0.3. The samples having the OD values of 0.3 or more were selected (FIG. 15).

3.2 HA Genomic DNA PCR

If the HA gene is located in the T DNA region of the transformed Agrobacterium rhizogenes, the HA gene would be integrated into the chromosome of the transgenic plant. Therefore, genomic DNA PCR was performed to confirm the presence of HA gene in transgenic hairy roots having higher OD value, obtained from the result of HA Ag ELISA. The HA primer set prepared in the first cloning step was used to perform general PCR. A HA band of 1,695 bp was found in the HA transgenic hairy root. Consequently, it can be seen that the HA gene was integrated into the chromosome of the hairy root (FIG. 16).

3.3 RT-PCR Analysis of HA and NA Transgenic Hairy Roots

An RT-PCR analysis was performed to confirm transcription of HA and NA genes in the transformants. As shown in FIG. 17 a, a specific primer set was designed. RNA was extracted using a Trireagent from the plant having higher OD value, obtained from the result of HA Ag ELISA, and cDNA was prepared. Then, PCR was performed using the primer set to amplify the 398˜680 region of HA1 (SEQ ID NOs. 14 and 15) at 52° C. for 35 cycles. Further, the RT-PCR analysis was performed to confirm the transcription of NA gene in the transformant. As shown in FIG. 17 b, a specific primer set (SEQ ID NOs. 16 and 17) was constructed. RNA was extracted from NA transgenic hairy root, and cDNA was prepared. Then, PCR was performed using the primer to amplify the 398˜680 region of NA (SEQ ID NOs. 16 and 17) at 52° C. for 35 cycles.

As shown FIG. 18 a, specific bands corresponding to 282 bp were detected in all of the HA transformants, and no band was observed in the empty transformant. It can be seen that the band strength depends on the amount of message. As shown FIG. 18 b, specific bands corresponding to 282 bp were detected in all of the NA transformants, and no band was observed in the hairy root infected with bacterium, which was not transformed. It can be seen that the band strength depends on the amount of message.

3.4 Production of HA and NA Antibodies in Rabbit

To produce antibodies used for the detection of HA and NA expression in Western blot and ELISA, a bacterial expression system was used to express HA and NA proteins. First, the sets of primers were constructed for cloning (see SEQ ID NOs. 18 to 21). HA was cloned into pET32a, NA into pGEX-GST4T-1, and expressed in BL21(DE3). The expression of HA and NA was detected at 83 kDa and 70 kDa, respectively. The expression condition was at an IPTG concentration of 0.5˜1 mM and a temperature of 37° C. for 3 hrs.

In order to confirm whether the expressed proteins were HA and NA, Western blot was performed using sera from chickens infected with avian influenza for the detection of HA expression, and using His antibody for the detection of NA expression. The pET vector has His-tags at both ends. Thus, HA was purified using an Ni-NTA agarose (Quiagen, Valencia, USA) (FIG. 19 a). NA was also purified using the Ni-NTA agarose by adding His sequence to the primer sequence (FIG. 19 b). Each of two rabbits was immunized with 23.5 μg for 10 weeks, and sera were collected from the rabbits. The collected sera were used for Western blot to determine the HA protein that was expressed in the transgenic plant (see FIGS. 20 and 21).

3.5 Western Blot for HA Protein Expression

Western blot was performed to confirm the correlation between expression levels of transcripts and proteins. As a positive control, the HA protein expressed in bacteria was used, and determined using polyclonal anti-HA antibody and monoclonal anti-HA antibody. Samples were prepared in the same manner as in the Ag ELISA. 52 μg of protein was loaded onto 10% SDS-PAGE gel, and run to a stacking gel at 100 V using a Tris-glycine buffer. Then, a resolving gel was subjected to electrophoresis at 200 V. The separated proteins were transferred to a PVDF membrane using a Mini-Trans-Blot electrophoretic transfer cell (Bio-rad, Hercules, USA) at 100 V for 1 hr. The membrane was blocked using 5% skim milk-TBST (TBS buffer plus 0.05% tween 20) at room normal temperature for 3 hrs, and slightly washed with 0.05% TBST. Then, the membrane was incubated with rabbit polyclonal anti-HA antibody (poly HA Ab) or mouse monoclonal anti-HA antibody at 1:4,000 dilution (2% skim milk in 0.2% TBST) and 1:2,000 dilution (2% skim milk in 0.2% TBST) at 4° C. overnight. Then, after washing for 15 min three times, the membrane was probed with horseradish peroxidase (HRP) conjugated rabbit anti-mouse-IgG or HRP-conjugated goat anti-rabbit IgG at a dilution of 1:2,000 (2% skim milk in 0.2% TBST) and 1:10,000 (2% skim milk in 0.2% TBST) at room temperature for 2 hrs. The membrane was washed with 0.05% TBST for 15 min three times, and then visualized using an ECL solution.

The hairy root transformed with HA gene was found to have a specific band, which was not detected in transformant with mock DNA. The result was the same as results of using other different types of antibodies (FIG. 20). The expression level in each lane was unique, which is similar pattern to the result of RT-PCR. Namely, the transformant HA12, HA8, and HA20 were found to show higher expression levels of HA protein. In order to determine immunoactivity by an animal test, the transformant HA12, HA8, and HA20 were freeze-dried and ground into powder. Western blot was performed to confirm the expression of HA protein in the powder. In the case of using polyclonal anti-HA antibody and monoclonal anti-HA antibody, the specific bands were detected in all of the samples. The transformant H20 showed higher expression level than HA20, thereby being used for the animal test (FIG. 21).

4. Glycosylation Pattern Analysis

In the Western blot, the specific band was always found to have a bigger size than the expected size of 63.5 kD, which was confirmed by glycosylation pattern analysis. 20 μg of protein was subjected to deglycosylation using PNGase F (New England BioLabs, Beverly, USA), and subjected to Western blot using polyclonal anti-HA antibody. As a result, the band size was found to be the same as the expected size (FIG. 22).

5. Establishment of Liquid Culture Condition for Mass-Production of Hairy Root

From the result of Western blot analysis in Example 3.5, it can be seen that the air-dried transgenic hairy root, H20 showed higher expression level of HA protein. Accordingly, for the mass-production of hairy root H20, the optimization of liquid culture condition was performed.

5.1 Selection of Optimal Medium for Mass-Production of Hairy Root HA-20

In order to select optimal media among MS (Murashige-Skoog) and B5, SH (Schenk-Hildebrandt), G/D, N/N, WPM and White media, which are used for tissue culture in the mass-production system, 20 ml of each media was put into a 300 ml flask, and 0.8 g of hairy root H20 was inoculated and cultured for 10 days. As shown in FIG. 23, the hairy root was found to be produced in MS and B5 media in a high yield. The composition of each medium is shown in the following Table 2.

TABLE 2 Medium Ingredients mg/L Gamborg's B-5 basal Ammonium sulfate 134 salt mixture Boric acid 3 Calcium chloride 113.24 anhydrous Cobalt chloride 6H₂O 0.025 Cupric sulfate 5H₂O 0.025 Na2-EDTA 37.3 Ferrous sulfate 7H₂O 27.8 Magnesium sulfate 122.09 Manganese sulfate H₂O 10 Molybdic acid (sodium 0.25 salt) 2H₂O Potassium iodide 0.75 Potassium nitrate 2500 Sodium phosphate 130.5 monobasic Zinc sulfate 7H₂O 2 Schenk and Ammonium phosphate 300 Hildebrandt(SH) Basal monobasic Salt Mixture Boric acid 5 Calcium chloride 151 anhydrous Cobalt chloride 6H₂O 0.1 Cupric sulfate 5H₂O 0.2 Na2-EDTA 20 Ferrous sulfate 7H₂O 15 Magnesium sulfate 195.4 Manganese sulfate H₂O 10 Molybdic acid (sodium 0.1 salt) 2H₂O Potassium iodide 1 Potassium nitrate 2500 Zinc sulfate 7H₂O 1 McCown's Woody Ammonium nitrate 400 Plant(WP) Basal Salt Boric acid 6.2 Mixture Calcium chloride 72.5 anhydrous Boric acid Calcium nitrate 386 Cupric sulfate 5H₂O 0.25 Na2-EDTA 37.3 Magnesium sulfate 180.7 Manganese sulfate H₂O 22.3 Molybdic acid (sodium 0.25 salt) 2H₂O Potassium phosphate 170 monobasic Potassium sulfate 990 Zinc sulfate 7H₂O 8.6 White's basal Boric acid 1.5 salt mixture Calcium nitrate 200 Ferric sulfate 2.5 Magnesium sulfate 360 Manganese sulfate 5.04 H₂O Potassium chloride 65 Potassium iodide 0.75 Potassium nitrate 80 Sodium phosphate 16.5 monobasic Sodium sulfate 200 Zinc sulfate 2.67 GRESSHOFF & DOY Ammonium Nitrate 1000 BASAL MEDIUM Boric Acid 0.3 Calcium Nitrate 241.2 Cobalt Chloride•6H₂O 0.025 Cupric Sulfate•5H₂O 0.025 Na2 EDTA•2H₂O 37.25 Ferrous Sulfate•7H₂O 27.85 Magnesium Sulfate, 17.099 Anhydrous Manganese Sulfate•H₂O 1 Molybdic Acid (Sodium 0.025 Salt)•2H₂O Potassium Chloride 65 Potassium Iodide 0.8 Potassium Nitrate 1000 Potassium Phosphate, 300 Monobasic Zinc Sulfate•7H₂O 0.3 D-Biotin 0.2 Glycine (Free Base) 4 myo-Inositol 10 Nicotinic Acid (Free 0.1 Acid) Pyridoxine•HCl 0.1 Thiamine•HCl 1 NITSCH & NITSCH Ammonium Nitrate 720 BASAL MEDIUM Boric Acid 10 w/VITAMINS Calcium Chloride, 166 Anhydrous Cupric Sulfate•5H₂O 0.025 Na2 EDTA•2H₂O 37.26 Ferrous Sulfate•7H₂O 27.8 Magnesium Sulfate, 90.372 Anhydrous Manganese Sulfate•H₂O 18.9 Molybdic Acid (Sodium 0.25 Salt)•2H₂O Potassium Nitrate 950 Potassium Phosphate, 68 Monobasic Zinc Sulfate•7H₂O 10 D-Biotin 0.05 Folic Acid 0.5 Glycine (Free Base) 2 myo-Inositol 100 Nicotinic Acid (Free 5 Acid) Pyridoxine•HCl 0.5 Thiamine•HCl 0.5

5.2 Optimum pH Level

In order to determine the optimum pH condition required for mass-culture, the yield in ½MS and MS media was examined according to each pH level to establish the optimum pH condition. 20 ml of each media was put into a 300 ml flask, 0.8 g of hairy root H20 was inoculated, and cultured at a known optimum pH 5.8 and, at each different pH 5.0, 7.0, and 8.0 for 10 days. The yield was higher at pH 8.0 than at the known optimum pH 5.8. Accordingly, pH 8.0 was selected as the optimum pH condition for mass-production (FIG. 24).

5.3 Selection of Optimal Sucrose Concentration

To determine the optimal concentration of sucrose used as an energy source in media ingredients, MS media was treated with various concentrations of sucrose such as 0 g/L, 7.5 g/L, 15 g/L, 30 g/L, and 60 g/L, and then the hairy roots were cultured in the liquid media. The yield of the hairy root was examined to determine the optimal concentration of sucrose. It was found that there was no difference in yield between the concentrations of 15 g/L and 30 g/L. Accordingly, the concentration of 15 g/L was determined as the optimal concentration to minimize production cost (FIG. 25).

5.4 Applicability of Selected Liquid Culture Condition to Mass-Production System

80 g of HA hairy root were inoculated in an 18 L bioreactor containing MS media (pH 8.0), and then cultured at 24° C. under a fluorescent light (500 or less Lux, 10 hrs light/14 hrs dark) for 14 days. The culture condition selected in Examples 5.1 to 5.3 was applied to an 18 L mass production system. As a result, the yield was increased from 80 g to 913 g to confirm the possibility of mass-production (FIG. 26).

6. Expression Level of HA Protein

In order to examine the expression level of HA in hairy root, an Ag ELISA was performed. HA protein expressed in bacteria was used as a positive control, and the HA expression level was measured using the above mentioned HA Ag ELISA.

First, proteins were extracted from 50 mg of freeze-dried hairy root using an extraction buffer at 4° C. for 3 hrs, and then immediately centrifuged at 10,000×g. A total volume was adjusted to 800 μl using the extraction buffer. For analysis, 100 μl of 2-fold serial dilutions were used. The HA protein expressed in bacteria was subjected to Coomassie staining to determine its concentration. 1.56 μl of bacterial recombinant HA protein was subjected to 2-fold serial dilutions with the extraction buffer, and loaded in a plate to perform the HA Ag ELISA. The Ag ELISA was performed in the same manner as in 3.1, and all samples were performed twice.

Based on the sample extracted from the air-dried sample, the standard OD value of trangenic—>expressed HA protein, of which concentration was known, was substituted with the OD value measured in the mock transgenic hairy root to calculate its concentration. Then, background was subtracted from the OD value of HA transgenic hairy root. That is, 1.173 μg calculated from the mock transgenic hairy root was subtracted from 2.103 μg calculated from HA transgenic hairy root to give 0.93 μg. 100 μl was used from the total 800 μl of extract, and thus 50 mg of transgenic hairy root contained 7.44 μg of HA protein. Accordingly, it can be seen that the hairy root contains the HA protein of 0.15 μg/mg as dry weight. Its content in water-soluble proteins was quantified to give 0.2% expression (FIG. 27).

7. Animal Test of HA

In vivo test was performed with the hairy root, of which HA expression was confirmed above. 6˜8-week-old female BALB/c mice were purchased, and four mice were maintained in individual cages, and their blood was collected at 1 week before performing immunization. The freeze-dried hairy root was orally administered to mice. In brief, mice were first fasted for 8 hrs or more with free access to water. At 30 min before immunization, 100 μl of 1.5% sodium bicarbonate was orally administered. Each of 1 mg and 5 mg of freeze-dried hairy root was diluted with 350 mM sodium bicarbonate to a total volume of 100 μl, and orally administered to two mice in a wild-type hairy root group and two mice in the transgenic hairy root group using a zonde needle Immunization was performed at 7-day intervals, and serum was isolated on day 8 after the last immunization. Feces were collected on day 3 after the last immunization (FIG. 28).

8. HA-Specific Antibody ELISA 8.1 Serum IgG ELISA

ELISA was performed to detect HA-specific antibody from the serum samples obtained after immunization. First, an ELISA plate was coated with the protein expressed in bacteria, and then the serum sample was added as a primary antibody. As a secondary antibody, anti-mouse IgG HRP was used. In this connection, the plate was coated with the protein in a concentration of 3 μg/ml, and the sample was diluted at a ratio of 1:50. The secondary antibody was diluted at a ratio of 1:2,000. As shown in the figure, it was found that in the group treated with HA transgenic hairy root, the HA-specific antibody was more highly induced than in the group treated with mock transgenic hairy root. There was no significant difference between the group treated with 5 mg and a negative group (FIG. 29).

8.2 Fecal IgA ELISA

100 mg of feces were suspended with 1 ml of extraction buffer (PBS-0.01% sodium azide with general protease inhibitor cocktail and 5% FBS) to prepare fecal samples. The suspension was vortexed at 4° C. for 30 min, and then centrifuged at 4° C. and 16,000×g. The supernatant was collected, and immediately subjected to ELISA. The supernatant was loaded without dilution, and the process was performed in the same manner as mentioned above. As a secondary antibody, an HRP-anti-mouse IgA was used at a 1:2,000 dilution. As shown in FIG. 30, in the group treated with 1 mg of HA transgenic hairy root, the induction of HA-specific antibody was about twice as high as the control group. 

1. A vector for plant transformation, pBSNB-TEV as shown in FIG.
 4. 2. A method for producing a recombinant foreign protein in a plant, comprising the step of introducing a nucleic acid molecule encoding the foreign protein into the vector of claim 1 to transform the plant.
 3. The method according to claim 2, wherein the transformation is performed using Agrobacterium comprising the vector for plant transformation, pBSNB-TEV as shown in FIG.
 4. 4. The method according to claim 2, wherein the plant is selected from the group consisting of rice (Oryza sativa), barley (Hordeum sativum), wheat (Triticum aestivum or Triticum vulgare), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), cucumber (Cucumis sativus), and Korean melon (Cucumis melo).
 5. The method according to claim 4, wherein the plant is Korean melon.
 6. The method according to claim 5, wherein the plant is a hairy root of Korean melon.
 7. The method of claim 2, wherein the foreign protein is a pathogenic antigen protein.
 8. The method according to claim 7, wherein the antigen protein is a surface protein of avian influenza virus.
 9. The method according to claim 8, wherein the surface protein is hemagglutinin (HA) or neuraminidase (NA).
 10. The method according to claim 7, wherein the antigen protein is a nucleoprotein (NP), an RNA polymerase protein PB2 (polymerase basic protein 2), or a polymerase acid protein (PA) of avian influenza virus.
 11. A vector for plant transformation, comprising a nucleic acid molecule encoding a surface protein of avian influenza virus, HA (hemagglutinin) or NA (neuraminidase) in a polycloning site of the vector of claim
 1. 12. A plant transformed with the vector of claim
 11. 13. The plant according to claim 12, wherein the plant is a hairy root of plant selected from the group consisting of rice (Oryza sativa), barley (Hordum sativum), wheat (Triticum aestivum or Triticum vulgare), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), cucumber (Cucumis sativus), and Korean melon (Cucumis melo).
 14. An oral avian influenza vaccine comprising the plant according to claim
 13. 15. The oral vaccine according to claim 14, wherein the plant is an air-dried hairy root of Korean melon.
 16. A method for mass-producing the plant of claim 13, wherein the plant is liquid-cultured in MS or B5 medium at pH 7 to
 8. 17. The method according to claim 16, wherein the medium contains sucrose in a concentration of 10 to 20 g/L.
 18. A recombinant foreign protein isolated from the plant of claim
 12. 19. The recombinant foreign protein according to claim 18, wherein the foreign protein is a surface protein of avian influenza virus, HA or NA.
 20. A method for preparing a reagent for diagnosing avian influenza virus infection, comprising the step of isolating a recombinant protein from the transgenic plant of claim
 12. 